RESUMO
Lignin is the most important natural source of aromatic compounds. The valorisation of lignin into aromatics requires fractionation steps that can be catalysed by ligninolytic enzymes. However, one of the main limitations of biological lignin fractionation is the low efficiency of biocatalysts; it is therefore crucial to enhance or to identify new ligninolytic enzymes. Currently, the screening of ligninolytic activities on lignin polymers represents a technological bottenleck and hinders the characterization and the discovery of efficient ligninolytic biocatalysts. An efficient and fast method for the measurement of such enzymatic activities is therefore required. In this work, we present a new electrochemical tool based on lignin-coated paper electrodes for the detection and the characterization of ligninolytic activity. The suitability of this method is demonstrated using a catalase-peroxidase isolated from Thermobacillus xylanilyticus.
Assuntos
Lignina , Peroxidases , Lacase , Lignina/química , Peroxidase , Compostos Orgânicos/químicaRESUMO
Lignocellulosic biomass is rich in lignins, which represent a bottomless natural source of aromatic compounds. Due to the high chemical complexity of these aromatic polymers, their biological fractionation remains challenging for biorefinery. The production of aromatics from the biological valorization of lignins requires the action of ligninolytic peroxidases and laccases produced by fungi and bacteria. Therefore, identification of efficient ligninolytic enzymes with high stability represents a promising route for lignins biorefining. Our strategy consists in exploiting the enzymatic potential of the thermophilic bacterium Thermobacillus xylanilyticus to produce robust and thermostable ligninolytic enzymes. In this context, a gene encoding a putative catalase-peroxidase was identified from the bacterial genome. The present work describes the production of the recombinant protein, its biochemical characterization, and ligninolytic potential. Our results show that the catalase-peroxidase from T. xylanilyticus is thermostable and exhibits catalase-peroxidase and manganese peroxidase activities. The electrochemical characterization using intermittent pulse amperometry showed the ability of the enzyme to oxidize small aromatic compounds derived from lignins. This promising methodology allows the fast screening of the catalase-peroxidase activity towards small phenolic molecules, suggesting its potential role in lignin transformation. KEY POINTS: ⢠Production and characterization of a new thermostable bacterial catalase-peroxidase ⢠The enzyme is able to oxidize many phenolic monomers derived from lignins ⢠Intermittent pulse amperometry is promising to screen ligninolytic enzyme.
Assuntos
Lignina , Peroxidase , Lignina/metabolismo , Catalase , Peroxidases/genética , Peroxidases/metabolismo , FenóisRESUMO
Aliphatic synthetic intermediates with high added value are generally produced from alkane sources (e.g., petroleum) by inert carbon-hydrogen (C-H) bond activation using classical chemical methods (i.e. high temperature, rare metals). As an alternative approach for these reactions, alkane monooxygenase from Pseudomonas putida (alkB) is able to catalyze the difficult terminal oxyfunctionalization of alkanes selectively and under mild conditions. Herein, we report an electrosynthetic system using an alkB biocathode which produces alcohols, epoxides, and sulfoxides through bioelectrochemical hydroxylation, epoxidation, sulfoxidation, and demethylation. The capacity of the alkB binding pocket to protect internal functional groups is also demonstrated. By coupling our alkB biocathode with a hydrogenase bioanode and using H2 as a clean fuel source, we have developed and characterized a series of enzymatic fuel cells capable of oxyfunctionalization while simultaneously producing electricity.
Assuntos
Alcanos/metabolismo , Fontes de Energia Bioelétrica/microbiologia , Oxigenases de Função Mista/metabolismo , Eletrodos , Transporte de Elétrons , Compostos de Epóxi/química , Hidroxilação , Metilação , Oxigênio/química , Pseudomonas putida/enzimologia , Safrol/análogos & derivados , Safrol/química , Especificidade por SubstratoRESUMO
Electrochemical ethanol oxidation was performed at an innovative hybrid architecture electrode containing TEMPO-modified linear poly(ethylenimine) (LPEI) and oxalate oxidase (OxOx) immobilized on carboxylated multi-walled carbon nanotubes (MWCNT-COOH). On the basis of chromatographic results, the catalytic hybrid electrode system completely oxidized ethanol to CO2 after 12â¯h of electrolysis. The fact that the developed system can catalyze ethanol electrooxidation at a carbon electrode confirms that organic oxidation catalysts combined with enzymatic catalysts allow up to 12 electrons to be collected per fuel molecule. The Faradaic efficiency of the hybrid system investigated herein lies above 87%. The combination of OxOx with TEMPO-LPEI to obtain a novel hybrid anode to oxidize ethanol to carbon dioxide constitutes a simple methodology with useful application in the development of enzymatic biofuel cells.
Assuntos
Eletrólise , Etanol/química , Dióxido de Carbono/química , Catálise , Óxidos N-Cíclicos/química , Eletrodos , Eletrólise/métodos , Enzimas Imobilizadas/química , Nanotubos de Carbono/química , Oxirredução , Oxirredutases/química , Polietilenoimina/químicaRESUMO
Enzymatic bioelectrocatalysis often requires an artificial redox mediator to observe significant electron transfer rates. The use of such mediators can add a substantial overpotential and obfuscate the protein's native kinetics, which limits the voltage of a biofuel cell and alters the analytical performance of biosensors. Herein, we describe a material for facilitating direct electrochemical communication with redox proteins based on a novel pyrene-modified linear poly(ethyleneimine). This method was applied for promoting direct bioelectrocatalytic reduction of O2 by laccase and, by immobilizing the catalytic subunit of nitrogenase (MoFe protein), to demonstrate the ATP-independent direct electroenzymatic reduction of N2 to NH3.
RESUMO
Increasing greenhouse gas emissions have resulted in greater motivation to find novel carbon dioxide (CO2 ) reduction technologies, where the reduction of CO2 to valuable chemical commodities is desirable. Molybdenum-dependent formate dehydrogenase (Mo-FDH) from Escherichia coli is a metalloenzyme that is able to interconvert formate and CO2 . We describe a low-potential redox polymer, synthesized by a facile method, that contains cobaltocene (grafted to poly(allylamine), Cc-PAA) to simultaneously mediate electrons to Mo-FDH and immobilize Mo-FDH at the surface of a carbon electrode. The resulting bioelectrode reduces CO2 to formate with a high Faradaic efficiency of 99±5 % at a mild applied potential of -0.66â V vs. SHE.
RESUMO
Over the past decade, there has been significant research in electrochemical reduction of CO2, but it has been difficult to develop catalysts capable of C-C bond formation. Here, we report bioelectrocatalysis of vanadium nitrogenase from Azotobacter vinelandii, where cobaltocenium derivatives transfer electrons to the catalytic VFe protein, independent of ATP-hydrolysis. In this bioelectrochemical system, CO2 is reduced to ethylene (C2H4) and propene (C3H6), by a single metalloenzyme.
RESUMO
An enzymatic electrosynthesis system was created by combining an aldehyde deformylating oxygenase (ADO) from cyanobacteria that catalyzes the decarbonylation of fatty aldehydes to alkanes and formic acid with an electrochemical interface. This system is able to produce a range of alkanes (octane to propane) from aldehydes and alcohols. The combination of this bioelectrochemical system with a hydrogenase bioanode yields a H2 /heptanal enzymatic fuel cell (EFC) able to simultaneously generate electrical energy with a maximum current density of 25â µA cm-2 at 0.6â V and produce hexane with a faradaic efficiency of 24 %.
RESUMO
The need to immobilize active enzyme, while ensuring high rates of substrate turnover and electronic charge transfer with an electrode, is a centrally important challenge in the field of bioelectrocatalysis. In this work, we demonstrate the use of confocal Raman microscopy as a tool for quantitation and molecular-scale structural characterization of ionomers and proteins within biocatalytic membranes to aid in the development of energy efficient biofuel cells. A set of recently available short side chain Aquivion ionomers spanning a range of equivalent weight (EW) suitable for enzyme immobilization was investigated. Aquivion ionomers (790 EW, 830 EW and 980 EW) received in the proton-exchanged (SO3H) form were treated with tetra-n-butylammonium bromide (TBAB) to neutralize the ionomer and expand the size of ionic domains for enzyme incorporation. Through the use of confocal Raman microscopy, membrane TBA+ ion content was predicted in calibration studies to within a few percent of the conventional titrimetric method across the full range of TBA+: SO3- ratios of practical interest (0.1 to 1.7). Protein incorporation into membranes was quantified at the levels expected in biofuel cell electrodes. Furthermore, features associated with the catalytically active, enzyme-coordinated copper center were evident between 400 and 500 cm-1 in spectra of laccase catalytic membranes, demonstrating the potential to interrogate mechanistic chemistry at the enzyme active site of biocathodes under fuel cell reaction conditions. When benchmarked against the 1100 EW Nafion ionomer in glucose/air enzymatic fuel cells (EFCs), EFCs with laccase air-breathing cathodes prepared from TBA+ modified Aquivion ionomers were able to reach maximum power densities (Pmax) up to 1.5 times higher than EFCs constructed with cathodes prepared from TBA+ modified Nafion. The improved performance of EFCs containing the short side chain Aquivion ionomers relative to Nafion is traced to effects of ionomer ion-exchange capacity (IEC, where IEC = EW-1), where the greater density of SO3- moieties in the Aquivion materials produces an environment more favorable to mass transport and higher TBA+ concentrations.
Assuntos
Fontes de Energia Bioelétrica , Técnicas Eletroquímicas , Lacase/análise , Compostos de Amônio Quaternário/análise , Biocatálise , Eletrodos , Íons/análise , Lacase/metabolismo , Microscopia Confocal , Compostos de Amônio Quaternário/metabolismo , Análise Espectral RamanRESUMO
The development of a hybrid, tri-catalytic architecture is demonstrated by immobilizing MWCNTs, TEMPO-modified linear poly(ethylenimine) and oxalate decarboxylase on an electrode to enable enhanced electrochemical oxidation of glycerol. This immobilized, hybrid catalytic motif results in a synergistic 3.3-fold enhancement of glycerol oxidation and collects up to 14 electrons per molecule of glycerol.
RESUMO
Nitrogenase, the only enzyme known to be able to reduce dinitrogen (N2) to ammonia (NH3), is irreversibly damaged upon exposure to molecular oxygen (O2). Several microbes, however, are able to grow aerobically and diazotrophically (fixing N2 to grow) while containing functional nitrogenase. The obligate aerobic diazotroph, Azotobacter vinelandii, employs a multitude of protective mechanisms to preserve nitrogenase activity, including a "conformational switch" protein (FeSII, or "Shethna") that reversibly locks nitrogenase into a multicomponent protective complex upon exposure to low concentrations of O2. We demonstrate in vitro that nitrogenase can be oxidatively damaged under anoxic conditions and that the aforementioned conformational switch can protect nitrogenase from such damage, confirming that the conformational change in the protecting protein can be achieved solely by regulating the potential of its [2Fe-2S] cluster. We further demonstrate that this protective complex preserves nitrogenase activity upon exposure to air. Finally, this protective FeSII protein was incorporated into an O2-tolerant bioelectrosynthetic cell whereby NH3 was produced using air as a substrate, marking a significant step forward in overcoming the crippling limitation of nitrogenase's sensitivity toward O2.
Assuntos
Azotobacter vinelandii/enzimologia , Nitrogenase/química , Oxigênio/metabolismo , Conformação Molecular , OxirreduçãoRESUMO
Diaphorase and a benzylpropylviologen redox polymer were combined to create a bioelectrode that can both oxidize NADH and reduce NAD+. We demonstrate how bioelectrocatalytic NAD+/NADH inter-conversion can transform a glucose/O2 enzymatic fuel cell (EFC) with an open circuit potential (OCP) of 1.1 V into an enzymatic redox flow battery (ERFB), which can be rapidly recharged by operation as an EFC.
RESUMO
Glycerol is a common fuel considered for bioenergy applications. Computational docking studies were performed on formate dehydrogenase from Candida boidinii (cbFDH) that showed that mesoxalate can bind to the buried active site of the holo form predicting that mesoxalate, a byproduct of glycerol oxidation, may act as its substrate. Spectroscopic assays and characterization by HPLC and GC/TCD have shown for the first time that cbFDH can act as a decarboxylase with mesoxalate. From this assessment, cbFDH was combined with NH2-TEMPO to form a novel hybrid anode to oxidize glycerol to carbon dioxide at near-neutral pH.
Assuntos
Candida/enzimologia , Técnicas Eletroquímicas , Formiato Desidrogenases/metabolismo , Glicerol/química , Dióxido de Carbono/química , Eletrodos , Formiato Desidrogenases/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estrutura Molecular , OxirreduçãoRESUMO
Nitrogenases are the only enzymes known to reduce molecular nitrogen (N2 ) to ammonia (NH3 ). By using methyl viologen (N,N'-dimethyl-4,4'-bipyridinium) to shuttle electrons to nitrogenase, N2 reduction to NH3 can be mediated at an electrode surface. The coupling of this nitrogenase cathode with a bioanode that utilizes the enzyme hydrogenase to oxidize molecular hydrogen (H2 ) results in an enzymatic fuel cell (EFC) that is able to produce NH3 from H2 and N2 while simultaneously producing an electrical current. To demonstrate this, a charge of 60â mC was passed across H2 /N2 EFCs, which resulted in the formation of 286â nmol NH3 â mg-1 MoFe protein, corresponding to a Faradaic efficiency of 26.4 %.
RESUMO
The reversible inhibition of laccase by arsenite (As(3+)) and arsenate (As(5+)) is reported for the first time. Oxygen-reducing laccase bioelectrodes were found to be inhibited by both arsenic species for direct electron-transfer bioelectrodes (using anthracene functionalities for enzymatic orientation) and for mediated electron-transfer bioelectrodes [using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as an electron mediator]. Both arsenic species were determined to behave via a mixed inhibition model (behaving closely to that of uncompetitive inhibitors) when evaluated spectrophotometrically using ABTS as the electron donor. Finally, laccase bioelectrodes were employed within an enzymatic fuel cell, yielding a self-powered biosensor for arsenite and arsenate. This conceptual self-powered arsenic biosensor demonstrated limits of detection (LODs) of 13 µM for arsenite and 132 µM for arsenate. Further, this device possessed sensitivities of 0.91 ± 0.07 mV/mM for arsenite and 0.98 ± 0.02 mV/mM for arsenate.
Assuntos
Arseniatos/farmacologia , Arsenitos/farmacologia , Técnicas Biossensoriais , Lacase/antagonistas & inibidores , Espectrofotometria UltravioletaRESUMO
Enzymatic biofuel cells are bioelectronic devices that utilize oxidoreductase enzymes to catalyze the conversion of chemical energy into electrical energy. This review details the advancements in the field of enzymatic biofuel cells over the last 30 years. These advancements include strategies for improving operational stability and electrochemical performance, as well as device fabrication for a variety of applications, including implantable biofuel cells and self-powered sensors. It also discusses the current scientific and engineering challenges in the field that will need to be addressed in the future for commercial viability of the technology.
Assuntos
Fontes de Energia Bioelétrica , Técnicas Biossensoriais/métodos , Eletroquímica/métodos , Enzimas Imobilizadas/química , Oxirredutases/químicaRESUMO
Isothermal titration calorimetry (ITC) may be used to determine the kinetic parameters of enzyme-catalyzed reactions when neither products nor reactants are spectrophotometrically visible and when the reaction products are unknown. We report here the use of the multiple injection method of ITC to characterize the catalytic properties of oxalate oxidase (OxOx) from Ceriporiopsis subvermispora (CsOxOx), a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin enzyme identified that catalyzes this reaction. The multiple injection ITC method of measuring OxOx activity involves continuous, real-time detection of the amount of heat generated (dQ) during catalysis, which is equal to the number of moles of product produced times the enthalpy of the reaction (ΔHapp). Steady-state kinetic constants using oxalate as the substrate determined by multiple injection ITC are comparable to those obtained by a continuous spectrophotometric assay in which H2O2 production is coupled to the horseradish peroxidase-catalyzed oxidation of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and by membrane inlet mass spectrometry. Additionally, we used multiple injection ITC to identify mesoxalate as a substrate for the CsOxOx-catalyzed reaction, with a kinetic parameters comparable to that of oxalate, and to identify a number of small molecule carboxylic acid compounds that also serve as substrates for the enzyme.
RESUMO
Electron mediation between NAD-dependent enzymes using quinone moieties typically requires the use of a diaphorase as an intermediary enzyme. The ability for a naphthoquinone redox polymer to independently oxidize enzymatically-generated NADH is demonstrated for application to glucose/O2 enzymatic fuel cells.
Assuntos
NAD/metabolismo , Naftoquinonas/química , Oxirredutases/metabolismo , Polímeros/química , Biocatálise , Técnicas Eletroquímicas , OxirreduçãoRESUMO
The complete electro-oxidation of glycerol to CO2 is performed through an oxidation cascade using a hybrid catalytic system combining a recombinant enzyme, oxalate decarboxylase from Bacillus subtilis, and an organic oxidation catalyst, 4-amino-TEMPO. This system is capable of electrochemically oxidizing glycerol at a carbon electrode collecting all 14 electrons per molecule.
Assuntos
Bacillus subtilis/enzimologia , Carboxiliases/metabolismo , Glicerol/metabolismo , Catálise , Óxidos N-Cíclicos/metabolismo , Técnicas Eletroquímicas , Eletrodos , Enzimas Imobilizadas/metabolismo , Modelos Moleculares , Oxirredução , Proteínas Recombinantes/metabolismoRESUMO
Enzymatic fuel cells (EFCs) are devices that can produce electrical energy by enzymatic oxidation of energy-dense fuels (such as glucose). When considering bioanode construction for EFCs, it is desirable to use a system with a low onset potential and high catalytic current density. While these two properties are typically mutually exclusive, merging these two properties will significantly enhance EFC performance. We present the rational design and preparation of an alternative naphthoquinone-based redox polymer hydrogel that is able to facilitate enzymatic glucose oxidation at low oxidation potentials while simultaneously producing high catalytic current densities. When coupled with an enzymatic biocathode, the resulting glucose/O2 EFC possessed an open-circuit potential of 0.864 ± 0.006 V, with an associated maximum current density of 5.4 ± 0.5 mA cm-2. Moreover, the EFC delivered its maximum power density (2.3 ± 0.2 mW cm-2) at a high operational potential of 0.55 V.